Pervasive selection for clinically relevant resistance and media adaptive mutations at very low antibiotic concentrations

Experimental evolution studies have shown that weak antibiotic selective pressures (i.e. when the antibiotic concentrations are far below the minimum inhibitory concentration, MIC) can select for resistant mutants, raising several unanswered questions. First, what are the lowest antibiotic concentrations at which selection for de novo resistance mutations can occur? Second, with weak antibiotic selections, which other types of adaptive mutations unrelated to the antibiotic selective pressure are concurrently enriched. Third, are the mutations selected under laboratory settings at sub-MIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at sub-MICs in presence of either of four clinically used antibiotics: fosfomycin, nitrofurantoin, tetracycline and ciprofloxacin. Antibiotic resistance evolution was investigated at concentrations ranging from 1/4th to 1/2000th of the MIC of the susceptible strain (MICsusceptible). Our results show that evolution was rapid across all the anitibiotics tested, and selection for fosfomycin and nitrofurantoin resistant mutants was observed at a concentration as low as 1/2000th of MICsusceptible. Several of the evolved resistant mutants showed increased growth yield and exponential growth rates, and outcompeted the susceptible ancestral strain in the absence of antibiotic as well, suggesting that adaptation to the growth environment occurred in parallel with selection for resistance. Genomic analysis of the resistant mutants showed that several of the mutations selected under these conditions are also found in clinical isolates, demonstrating that experimental evolution at very low antibiotic levels can help in identifying novel mutations that contribute to bacterial adaptation during sub-MIC exposure in real life settings.